Apparatus and Methods for Analyte Measurement and Immunoassay

ABSTRACT

The present invention relates to methods for measuring an amount of an analyte using an electrochemical assay in a conduit comprising a sensor, wherein said sensor comprises an electrode having a surface layer of immobilized antibody that binds said analyte, and a counter/reference electrode disposed within said conduit. A solution comprising a substrate for said enzyme and at least one air segment contacts the sensor to remove unbound analyte and labeled antibody from a region of the sensor. The disclosed invention is adaptable to the point-of-care clinical diagnostic field, including use in accident sites, emergency rooms, surgery, nursing homes, intensive care units, and non-medical environments.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional application of Ser. No. 10/087,730,filed Mar. 5, 2002, the entire contents of which is hereby incorporatedherein by reference.

FIELD OF THE INVENTION

In its broadest aspect, the invention relates to an apparatus and methodfor rapid in situ determinations of analytes in liquid samples that iscapable of being used, for example, in the point-of-care clinicaldiagnostic field, including use at accident sites, emergency rooms, insurgery, in intensive care units, and also in non-medical environments.

The present invention thus relates to an apparatus and its method of usefor determining the presence and/or concentrations of analytes in aliquid sample. More particularly, the invention provides single-usedisposable cartridges, adapted for conducting diverse real-time or nearreal-time assays of analytes. The invention further relates to acartridge that provides novel features for processing a metered portionof a sample. The cartridge incorporates novel features for precise andflexible control of the movement of a sample or a second fluid withinthe cartridge, and for the optional amending of sample or fluid with oneor more additional reagents or compounds during an assay. While thecartridges of the present invention are intended for use in a readingapparatus, they may also be used separately. They comprise conduits,pump means, a fluid, metering means, valves, and an optional sensor orsensors for determining the position or positions of liquids within theconduits. In specific embodiments, the invention relates to thedetermination of analytes in biological samples such as blood usingelectrochemical immunosensors or other ligand/ligand receptor-basedbiosensors. The invention further relates to a simplified constructionof a biosensor, in particular for fabrication of electrochemicalimmunoassay biosensors capable of determining a wide range of analytesfor which receptors or antibodies can be obtained.

BACKGROUND OF THE INVENTION

A multitude of laboratory tests for analytes of interest are performedon biological samples for diagnosis, screening, disease staging,forensic analysis, pregnancy testing, drug testing, and other reasons.While a few qualitative tests, such as pregnancy tests, have beenreduced to simple kits for the patient's home use, the majority ofquantitative tests still require the expertise of trained technicians ina laboratory setting using sophisticated instruments. Laboratory testingincreases the cost of analysis and delays the results. In manycircumstances, delay can be detrimental to a patient's condition orprognosis, such as for example the analysis of markers indicating ofmyocardial infarction. In these critical situations and others, it wouldbe advantageous to be able to perform such analyses at the point ofcare, accurately, inexpensively, and with a minimum of delay.

A disposable sensing device for measuring analytes in a sample of bloodis disclosed by Lauks in U.S. Pat. No. 5,096,669. Other devices aredisclosed by Davis, et al. in U.S. Pat. Nos. 5,628,961 and 5,447,440 fora clotting time. The disclosed apparatuses comprise reading apparatusand a cartridge which fits into the reading apparatus for the purpose ofmeasuring analyte concentrations and viscosity changes in a sample ofblood as a function of time. A potential problem with disposable devicesis variability of fluid test parameters from cartridge to cartridge dueto manufacturing tolerances or machine wear. Zelin, U.S. Pat. No.5,821,399 discloses methods to overcome this problem using automaticflow compensation controlled by a reading apparatus using conductimetricsensors located within a cartridge. U.S. Pat. Nos. 5,096,669, 5,628,961,5,447,440, and 5,821,399 are hereby incorporated in their respectiveentireties by reference.

Antibodies are extensively used in the analysis of biological analytes.For a review of basic principles see Eddowes, Biosensors 3:1-15, 1987.While in all such applications an antibody provides analyte bindingspecificity, a variety of different analytical approaches have beenemployed to detect, either directly or indirectly, the binding of anantibody to its analyte. Various alternative assay formats (other thanthose used in typical research laboratories, such as Western blotting)have been adopted for quantitative immunoassay, which are distinguishedfrom qualitative immunoassay kits, such as pregnancy testing kits. As anexample of antibody use, Ligler, in U.S. Pat. No. 5,183,740 disclosed aflow-through immunosensor device comprising a column loaded withparticles coated with an antibody bound to a labeled antigen. When asample is flowed through the column, unlabeled antigen displaces labeledantigen which then flows to a detector. In an alternative approach,Giaever, in U.S. Pat. No, 4,018,886 discloses the use of magneticparticles coated with an antibody, which are first magneticallycirculated in a sample to accelerate binding of the analyte, thenconcentrated in a small volume, and finally the antibody-antigen complexis cleaved from the bead to yield a concentrated solution of thecomplex. U.S. Pat. No. 5,073,484 to Swanson discloses a method in whicha fluid-permeable solid medium has reaction zones through which a sampleflows. A reactant that is capable of reaction with the analyte is boundto the solid medium in a zone. A localized, detectable product isproduced in the zone when analyte is present. In a similar concept, U.S.Pat. No. 5,807,752 to Brizgys discloses a test system in which a solidphase is impregnated with a receptor for an analyte of interest. Asecond analyte-binding partner attached to aspectroscopically-determinable label and a blocking agent is introduced,and the spatial distribution of the label is measured. Spectroscopicmeasurements require a light transducer, typically a photomultiplier,phototransistor, or photodiode, and associated optics that may be bulkyor expensive, and are not required in electrochemical methods, in whichan electrical signal is produced directly.

Because a quantitative immunoassay typically requires multiple steps(e.g., a binding step followed by a rinse step with a solution that mayor may not contain a second reagent), most of the foregoing methods areeither operated manually, or require bulky machinery with complexfluidics. An example of the latter approach is provided in U.S. Pat. No.5,201,851 which discloses methods providing complex fluidics for verysmall volumes on a planar surface. This method is used, for example, inthe Biacore system (Pharmacia) which is housed in a bench-top instrumentand uses surface plasmon resonance to detect binding of macromoleculesto an immobilized receptor on a surface. See, U.S. Pat. Nos. 5,242,828and 5,313,264.

The foregoing references disclose optical means for detecting thebinding of an analyte to a receptor. Electrochemical detection, in whichbinding of an analyte directly or indirectly causes a change in theactivity of an electroactive species adjacent to an electrode, has alsobeen applied to immunoassay. For a review of electrochemicalimmunoassay, see: Lauren, et al., Methods in Enzymology, vol. 73,“Electroimmunoassay”, Academic Press, New York, 339, 340, 346-348(1981). For example, U.S. Pat. No, 4,997,526 discloses a method fordetecting an analyte that is electroactive. An electrode poised at anappropriate electrochemical potential is coated with an antibody to theanalyte. When the electroactive analyte binds to the antibody, a currentflows at the electrode. This approach is restricted in the analytes thatcan be detected: only those analytes that have electrochemical midpointpotentials within a range that does not cause the electrode to performnon-specific oxidation or reduction of other species present in thesample by the electrode. The range of analytes that may be determined isextended by the method disclosed in U.S. Pat. No. 4,830,959, which isbased upon enzymatic conversion of a non-mediator to a mediator.Application of the aforementioned invention to sandwich immunoassays,where a second antibody is labeled with an enzyme capable of producingmediator from a suitable substrate, means that the method can be used todetermine electroinactive analytes.

Other electrical properties have also been employed in analyte sensors.U.S. Pat. Nos. 4,334,850 and 4,916,075 to Malmros disclose apolyacetylene film comprising an element whose electrical resistancevaries in response to the presence of an analyte. Electric field effectsare exploited in U.S. Pat. No, 4,238,757 to Schenck, where afield-effect transistor (FET) immunosensor is disclosed. An immunoassaybased upon the use of an analyte labeled with a particle that affectsthe electrical reactance of an electrode is disclosed by Pace in U.S.Pat. No, 4,233,144. It will be apparent from these descriptions, that ineach of the foregoing examples where other electrical properties areemployed, the existence or magnitude of the required electrical propertychange may be different for each analyte. Therefore, there exists a needfor assay techniques that can be automated and applied to diverseanalytes to create assays with substantially uniform characteristicsindependent of specific characteristics of individual analyte species.

Microfabrication techniques (e.g., photolithography and plasmadeposition) are attractive for construction of multilayered sensorstructures in confined spaces. Methods for microfabrication ofelectrochemical immunosensors, for example on silicon substrates, aredisclosed in U.S. Pat. No. 5,200,051 to Cozette, et al., which is herebyincorporated in its entirety by reference. These include dispensingmethods, methods for attaching biological reagent, e.g., antibodies, tosurfaces including photoformed layers and microparticle latexes, andmethods for performing electrochemical assays.

In an electrochemical immunosensor, the binding of an analyte to itscognate antibody produces a change in the activity of an electroactivespecies at an electrode that is poised at a suitable electrochemicalpotential to cause oxidation or reduction of the electroactive species.There are many arrangements for meeting these conditions. For example,electroactive species may be attached directly to an analyte (seeabove), or the antibody may be covalently attached to an enzyme thateither produces an electroactive species from an electroinactivesubstrate, or destroys an electroactive substrate. See, M. J. Green(1987) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 316:135-142, for areview of electrochemical immunosensors.

Therefore, there exists within the field of analyte sensing, and inparticular for applications in which analytes must be determined withinbiological samples such as blood, a need for apparatus that can rapidlyand simply determine analytes at the point-of-care, and can be performedby less highly trained staff than is possible for conventionallaboratory-based testing. Frequently, it would be of benefit in thediagnosis and treatment of critical medical conditions for the attendingphysician or nurse to be able to obtain clinical test results withoutdelay. Furthermore, an improved apparatus should be adaptable todetermination of a range of analytes and capable of single-use so thatimmediate disposal of the sample after testing minimizes the risk ofbiological or chemical contamination. These and other needs are met bythe present invention as will become clear to one of skill in the art towhich the invention pertains upon reading the following disclosure.

SUMMARY OF THE INVENTION

It is therefore an objective of the present invention to provideimproved apparatus and methods for the determination of analytes in aliquid sample, which avoid the aforementioned disadvantages anddrawbacks.

It is a further objective of the present invention to permit rapid,inexpensive, in situ determinations of analytes using a cartridge havingan array of analyte sensors and means for sequentially presenting asample and a fluid (amended or not) to the analyte array. The cartridgesare designed to be preferably operated with a reading device, such as,for example, disclosed in U.S. Pat. No. 5,096,669 to Lauks, et al.,issued Mar. 17, 1992, or U.S. Pat. No. 5,821,399 to Zelin, issued Oct.13, 1998, which are hereby incorporated in their respective entiretiesby reference.

The present invention provides cartridges, and methods for their use,for the processing of liquid samples to determine the presence or amountof an analyte within the sample. In specific embodiments the cartridgecontains a metering means, which permits an unmetered volume of a sampleto be introduced into a cartridge and from which a metered amount can beprocessed by the cartridge and its associated reading apparatus. Thusthe physician or operator is relieved of the task of accuratelymeasuring the volume of sample prior to measurement, with consequentsavings of time, effort, and also increased accuracy andreproducibility. In most specific embodiments, the metering meanscomprises an elongated sample chamber bounded by a capillary stop andhaving along its length an air entry point. Air pressure exerted at theair entry point drives a metered volume of the sample past the capillarystop. The metered volume is predetermined by the volume of the samplechamber between the air entry point and the capillary stop.

A cartridge according to the present invention has the advantage thatthe sample and a second fluid can contact the sensor array at differenttimes during an assay sequence. The sample and second fluid may also beindependently amended with other reagents or compounds present initiallyas dry coatings within the respective conduits. Controlled motion of theliquids within the cartridge further permits more than one substance tobe amended into each liquid whenever the sample or fluid is moved to anew region of the conduit. In this way, provision is made for multipleamendments to each fluid, greatly extending the complexity of automatedassays that can be performed, and therefore enhancing the utility of thepresent invention.

It is therefore an objective of the present invention to provide aflexible analyte analysis system, capable of adaptation to diverse assayprotocols. Control of liquid motion is achieved through coordinatedaction of pump means, valves, conduit restrictions, air segments, andconductimetric and other sensors. The cartridge is intended for use inconjunction with a reading device, which coordinates liquid movementswithin the cartridge. Pump means are provided that apply pressure todisplace sample and fluid through the conduits of the cartridge. Precisecontrol of the movement of the sample and fluid is provided in someembodiments by one or more conductimetric sensors disposed within theconduits, which sense the presence or absence of a conductive fluid atparticular points. This information is optionally used to control thepump means. In other embodiments, the cartridge further comprises valvesthat control the direction of sample and fluid movement. For example, inone embodiment a valve that closes after contact with a liquid enablesone pump means to move both the sample and a second liquid sequentiallyover the analyte sensor array. Furthermore, in some embodiments, meansare provided to introduce one or more air segments into the secondconduit to segment the liquid therein and thus prevent mixing betweensegments.

In a disposable cartridge, the amount of liquid contained is preferablykept small to minimize cost and size. Therefore, in the presentinvention, segments within the conduits are also used to assist incleaning and rinsing the conduits by passing the air-liquid interface ofa segment over the sensor array or other region to be rinsed at leastonce. It has been surprisingly found that more efficient rinsing, usingless fluid, is achieved by this method compared to continuous rinsing bya larger volume of fluid.

Restrictions within the conduits serve several purposes in the presentinvention. A capillary stop located between the sample chamber and firstconduit is used to prevent displacement of a sample introduced into theholding chamber until sufficient pressure is applied to overcome theresistance of the capillary stop. A restriction within the secondconduit is used to divert wash fluid along an alternative pathwaytowards the waste chamber when the fluid reaches the constriction. Smallholes in the gasket, together with a hydrophobic coating, are providedto prevent flow from the first conduit to the second conduit untilsufficient pressure is applied. Features that control the flow ofliquids within and between the conduits of the present invention areherein collectively termed “valves.” In these and other ways, thepresent invention has as an objective the provision of a flexible systemadaptable to diverse assays, as will become evident to one of skill inthe art upon reading the disclosure.

One embodiment of the invention, therefore, provides a single-usecartridge with a sample-holding chamber connected to a first conduitwhich contains an analyte sensor or array of analyte sensors. A secondconduit, partly containing a fluid, is connected to the first conduitand air segments can be introduced into the fluid in the second conduitin order to segment it. Pump means are provided to displace the samplewithin the first conduit, and displaces fluid from the second conduitinto the first conduit. Thus, the sensor or sensors can be contactedfirst by a sample and then by a second fluid.

A second embodiment of the cartridge includes a closeable valve locatedbetween the first conduit and a waste chamber. This embodiment permitsdisplacement of the fluid from the second conduit into the first conduitusing only a single pump means connected to the first conduit. Thisembodiment further permits efficient washing of the conduits of thecartridge of the present invention, which is an important feature of asmall single-use cartridge. In operation, the sample is displaced tocontact the sensors, and is then displaced through the closeable valveinto the waste chamber. Upon wetting, the closeable valve seals theopening to the waste chamber, providing an airtight seal that allowsfluid in the second conduit to be drawn into contact with the sensorsusing only the pump means connected to the first conduit. In thisembodiment, the closeable valve permits the fluid to be displaced inthis manner and prevents air from entering the first conduit from thewaste chamber.

In a third embodiment, both a closeable valve and means for introducingsegments into the conduit are provided. This embodiment has manyadvantages, among which is the ability to reciprocate a segmented fluidover the sensor or array of sensors. Thus a first segment or set ofsegments is used to rinse a sensor, and then a fresh segment replaces itfor taking measurements. Only one pump means (that connected to thefirst conduit) is required.

In a fourth embodiment, which is the preferred embodiment, analytemeasurements are performed in a thin-film of liquid coating an analytesensor. Such thin-film determinations are preferably performedamperometrically. The cartridge of the preferred embodiment differs fromthe foregoing embodiments in having both a closeable valve that issealed when the sample is expelled through the valve, and an air ventwithin the conduits that permits at least one air segment to besubsequently introduced into the measuring fluid, thereby increasing theefficiency with which the sample is rinsed from the sensor, and furtherpermitting removal of substantially all the liquid from the sensor priorto measurement, and still further permitting segments of fresh liquid tobe brought across the sensor to permit sequential, repetitivemeasurements for improved accuracy and internal checks ofreproducibility.

The analysis scheme for the detection of low concentrations ofimmunoactive analyte relies on the formation of an enzyme labeledantibody/analyte/surface-bound antibody “sandwich” complex. Theconcentration of analyte in a sample is converted into a proportionalsurface concentration of an enzyme. The enzyme is capable of amplifyingthe analyte's chemical signal by converting a substrate to a detectableproduct. For example, where alkaline phosphatase is the enzyme, a singleenzyme molecule can produce several thousand detectable molecules perminute, providing several orders of magnitude improvement in thedetectability of the analyte compared to schemes in which anelectroactive species is attached to the antibody in place of alkalinephosphatase.

In immunosensor embodiments, it is advantageous to contact the sensorfirst with a sample and then with a wash fluid prior to recording aresponse from the sensor. In specific embodiments, the sample is amendedwith an antibody-enzyme conjugate that binds to the analyte of interestwithin the sample before the amended sample contacts the sensor. Bindingreactions in the sample produce an analyte/antibody-enzyme complex. Thesensor comprises an immobilized antibody to the analyte, attached closeto an electrode surface. Upon contacting the sensor, theanalyte/antibody-enzyme complex binds to the immobilized antibody nearthe electrode surface. It is advantageous at this point to remove fromthe vicinity of the electrode as much of the unbound antibody-enzymeconjugate as possible to minimize background signal from the sensor. Theenzyme of the antibody-enzyme complex is advantageously capable ofconverting a substrate, provided in the fluid, to produce anelectrochemically active species. This active species is produced closeto the electrode and provides either a current from a redox reaction atthe electrode when a suitable potential is applied (amperometricoperation). Alternatively, if the electroactive species is an ion, itcan be measured potentiometrically. In amperometric measurements thepotential may either be fixed during the measurement, or variedaccording to a predetermined waveform. For example, a triangular wavecan be used to sweep the potential between limits, as is used in thewell-known technique of cyclic voltammetry. Alternatively, digitaltechniques such as square waves can be used to improve sensitivity indetection of the electroactive species adjacent to the electrode. Fromthe current or voltage measurement, the amount or presence of theanalyte in the sample is calculated. These and other analyticalelectrochemical methods are well known in the art.

In embodiments in which the cartridge comprises an immunosensor, theimmunosensor is advantageously microfabricated from a base sensor of anunreactive metal such as gold, platinum or iridium, which is overlaidwith a bioactive layer attached to a microparticle, for example latexparticles. The microparticles are dispensed onto the electrode surface,forming an adhered, porous bioactive layer. The bioactive layer has theproperty of binding specifically to the analyte of interest, or ofmanifesting a detectable change when the analyte is present, and is mostpreferably an immobilized antibody directed against the analyte.

In operation, therefore, one goal of the present invention is to providean immunosensor cartridge that is preferably operated in a basic senseas follows. (However, the invention is not restricted to embodimentscomprising an immunosensor, but includes any ligand-receptorinteraction.) An unmetered amount of a preferably biological sample isplaced into the sample chamber of the cartridge, and the cartridge isplaced into a reading apparatus. A metered portion of the sample isamended with at least one antibody-enzyme conjugate, and is thencontacted with the immunosensor. A second fluid, which contains anelectroinactive substrate for the enzyme, is used to rinse theimmunosensor substantially free of unbound antibody-enzyme conjugate,and the electrical response of the immunosensor electrode is recordedand analyzed for the presence, or amount of, the analyte of interest.The cartridge may contain a plurality of immunosensors and reagents.

After the reading, the operator removes and discards the cartridge. Thereader is then ready for another measurement. While the use of theinvention will frequently be referred to in a biological or medicalcontext, it will be appreciated that the present invention may bepracticed in any situation where it is desired to perform in situchemical analyses of liquid samples at speeds which approach real-time.

A further object of the invention is to provide a novel means of makingan electrochemical measurement in a conduit, whereby an immunosensor isexposed to sample and a fluid containing a substrate, after which thefluid is removed from the conduit except for a thin layer of fluid onthe wall of the conduit in the vicinity of the sensor.

While the invention is described in terms of an immunoassay cartridgeapplication, the invention is envisaged as containing within its scopeother clinical chemical assays known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objectives, features and advantages of the presentinvention are described in the following detailed description of thespecific embodiments and are illustrated in the following figures inwhich:

FIG. 1 is an isometric top view of an immunosensor cartridge cover.

FIG. 2 is an isometric bottom view of an immunosensor cartridge cover.

FIG. 3 is a top view of the layout of a tape gasket for an immunosensorcartridge.

FIG. 4 is an isometric top view of an immunosensor cartridge base.

FIG. 5 is a schematic view of the layout of an immunosensor cartridge.

FIG. 6 is a schematic view of the fluid and air paths within animmunosensor cartridge, including sites for amending fluids with dryreagents.

FIG. 7 illustrates the principle of operation of an electrochemicalimmunosensor.

FIG. 8 is a side view of the construction of an electrochemicalimmunosensor with antibody-labeled particles not drawn to scale.

FIG. 9 is a top view of the mask design for the conductimetric andimmunosensor electrodes for an immunosensor cartridge.

FIG. 10 illustrates the electrochemical responses of immunosensorsconstructed with an anti-HCG antibody when presented with 50 mU/mL, ofHCG.

FIG. 11 illustrates the electrochemical response (current versus time)of an immunosensor constructed with an anti-HCG antibody when presentedwith various amounts of HCG from 0 to 50 mU/mL.

FIG. 12 illustrates the maximum current obtained when an immunosensorconstructed with an anti-HCG antibody is presented with various amountsof HCG.

FIG. 13 is a schematic illustration of enzymatic regeneration of anelectroactive species.

FIG. 14 illustrates segment forming means.

FIG. 15 is a top view of the preferred embodiment of an immunosensorcartridge.

FIG. 16 is a schematic view of the fluidics of the preferred embodimentof an immunosensor cartridge.

FIG. 17 illustrates the electrochemical response (current versus time),and other responses, of a preferred embodiment of an immunosensor.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In Section A, a description of specific embodiments of the immunosensorof the present invention is provided together with three EXAMPLES oftheir use. In Section B, the preferred embodiment is described, togetherwith one EXAMPLE of its use.

A. Specific Embodiments

Cartridge Construction:

Referring to the figures, the cartridge of the present inventioncomprises a cover, FIGS. 1, 2, a base, FIG. 4, and a thin-film adhesivegasket, FIG. 3, disposed between the base and the cover. Referring nowto FIG. 1, the cover 1 is made of a rigid material, preferably plastic,capable of repetitive deformation at flexible hinge regions 5, 9, 10without cracking. The cover comprises a lid 2, attached to the main bodyof the cover by a flexible hinge 9. In operation, after introduction ofa sample into the sample holding chamber 34, the lid can be secured overthe entrance to the sample entry port 4, preventing sample leakage, andthe lid is held in place by hook 3. The cover further comprises twopaddles 6, 7, that are moveable relative to the body of the cover, andwhich are attached to it by flexible hinge regions 5, 10. In operation,when operated upon by a pump means, paddle 6 exerts a force upon an airbladder comprised of cavity 43, which is covered by thin-film gasket 21,to displace fluids within conduits of the cartridge. When operated by asecond pump means, paddle 7 exerts a force upon the gasket 21, which candeform because of slits 22 cut therein. The cartridge is adapted forinsertion into a reading apparatus, and therefore has a plurality ofmechanical and electrical connections for this purpose. It should alsobe apparent that manual operation of the cartridge is possible. Thus,upon insertion of the cartridge into a reading apparatus, the gaskettransmits pressure onto a fluid-containing foil pack filled withapproximately 130 uL of analysis/wash solution (“fluid”) located incavity 42, rupturing the package upon spike 38, and expelling fluid intoconduit 39, which is connected via a short transecting conduit in thebase to the sensor conduit. The analysis fluid fills the front of theanalysis conduit first pushing fluid onto a small opening in the tapegasket that acts as a capillary stop. Other motions of the analyzermechanism applied to the cartridge are used to inject one or moresegments into the analysis fluid at controlled positions within theanalysis conduit. These segments are used to help wash the sensorsurface and the surrounding conduit with a minimum of fluid.

The cover further comprises a hole covered by a thin pliable film 8. Inoperation, pressure exerted upon the film expels one or more airsegments into a conduit 20 through a small hole 28 in the gasket.

Referring to FIG. 2, the lower surface of the base further comprisessecond conduit 11, and first conduit 15. Second conduit 11 includes aconstriction 12, which controls fluid flow by providing resistance tothe flow of a fluid. Optional coatings 13, 14 provide hydrophobicsurfaces, which together with gasket holes 31, 32, control fluid flowbetween conduits 11, 15. A recess 17 in the base provides a pathway forair in conduit 34 to pass to conduit 34 through hole 27 in the gasket.

Referring to FIG. 3, thin-film gasket 21 comprises various holes andslits to facilitate transfer of fluid between conduits within the baseand the cover, and to allow the gasket to deform under pressure wherenecessary. Thus, hole 24 permits fluid to flow from conduit 11 intowaste chamber 44: hole 25 comprises a capillary stop between conduits 34and 11: hole 26 permits air to flow between recess 18 and conduit 40:hole 27 provides for air movement between recess 17 and conduit 34: andhole 28 permits fluid to flow from conduit 19 to waste chamber 44 viaoptional closeable valve 41. Holes 30 and 33 permit the plurality ofelectrodes that are housed within cutaways 35 and 37, respectively, tocontact fluid within conduit 15. In a specific embodiment, cutaway 37houses a ground electrode, and/or a counter-reference electrode, andcutaway 35 houses at least one analyte sensor and, optionally, aconductimetric sensor.

Referring to FIG. 4, conduit 34 is the sample holding chamber thatconnects the sample entry port 4 to first conduit 11 in the assembledcartridge. Cutaway 35 houses the analyte sensor or sensors, or ananalyte responsive surface, together with an optional conductimetricsensor or sensors. Cutaway 37 houses a ground electrode if needed as areturn current path for an electrochemical sensor, and may also house anoptional conductimetric sensor. Cutaway 36 provides a fluid path betweengasket holes 31 and 32 so that fluid can pass between the first andsecond conduits. Recess 42 houses a fluid-containing package, e.g., arupturable pouch, in the assembled cartridge that is pierced by spike 38because of pressure exerted upon paddle 7 upon insertion into a readingapparatus. Fluid from the pierced package flows into the second conduitat 39. An air bladder is comprised of recess 43 which is sealed on itsupper surface by gasket 21. The air bladder is one embodiment of a pumpmeans, and is actuated by pressure applied to paddle 6 which displacesair in conduit 40 and thereby displaces the sample from sample chamber34 into first conduit 15.

The location at which air enters the sample chamber (gasket hole 27)from the bladder, and the capillary stop 25, together define apredetermined volume of the sample chamber. An amount of the samplecorresponding to this volume is displaced into the first conduit whenpaddle 6 is depressed. This arrangement is therefore one possibleembodiment of a metering means for delivering a metered amount of anunmetered sample into the conduits of the cartridge.

In the present cartridge, a means for metering a sample segment isprovide in the base plastic part. The segment size is controlled by thesize of the compartment in the base and the position of the capillarystop and air pipe holes in the tape gasket. This volume can be readilyvaried from 2 to 200 microliters. Expansion of this range of samplesizes is possible within the context of the present invention.

The fluid is pushed through a pre-analytical conduit 11 that can be usedto amend a reagent (e.g., particles or soluble molecules) into thesample prior to its presentation at the sensor conduit 19.Alternatively, the amending reagent may be located in portion 15, beyondportion 16. Pushing the sample through the pre-analytical conduit alsoserves to introduce tension into the diaphragm pump paddle 7 whichimproves its responsiveness for actuation of fluid displacement.

In some assays, metering is advantageous if quantitation of the analyteis required. A waste chamber is provided, 44, for sample and/or fluidthat is expelled from the conduit, to prevent contamination of theoutside surfaces of the cartridge. A vent connecting the waste chamberto the external atmosphere is also provided, 45. A feature of thecartridge is that once a sample is loaded, analysis can be completed andthe cartridge discarded without the operator or others contacting thesample.

Referring now to FIG. 5, a schematic diagram of the features of acartridge and components is provided, wherein 51-57 are portions of theconduits and sample chamber that can optionally be coated with dryreagents to amend a sample or fluid. The sample or fluid is passed atleast once over the dry reagent to dissolve it. Reagents used to amendsamples or fluid within the cartridge include antibody-enzymeconjugates, or blocking agents that prevent either specific ornon-specific binding reactions among assay compounds. A surface coatingthat is not soluble but helps prevent non-specific adsorption of assaycomponents to the inner surfaces of the cartridges can also be provided.

Within a segment of sample or fluid, an amending substance can bepreferentially dissolved and concentrated within a predetermined regionof the segment. This is achieved through control of the position andmovement of the segment. Thus, for example, if only a portion of asegment, such as the leading edge, is reciprocated over the amendedsubstance, then a high local concentration of the substance can beachieved close to the leading edge. Alternatively, if an homogenousdistribution of the substance is desired, for example if a knownconcentration of an amending substance is required for a quantitativeanalysis, then further reciprocation of the sample or fluid will resultin mixing and an even distribution.

In specific embodiments, a closeable valve is provided between the firstconduit and the waste chamber. In one embodiment, this valve, 58, iscomprised of a dried sponge material that is coated with an impermeablesubstance. In operation, contacting the sponge material with the sampleor a fluid results in swelling of the sponge to fill the cavity 41,thereby substantially blocking further flow of liquid into the wastechamber 44. Furthermore, the wetted valve also blocks the flow of airbetween the first conduit and the waste chamber, which permits the firstpump means connected to the sample chamber to displace fluid within thesecond conduit, and to displace fluid from the second conduit into thefirst conduit in the following manner. After the sample is exposed tothe sensor for a controlled time, the sample is moved into thepost-analytical conduit 19 where it can be amended with another reagent.It can then be moved back to the sensor and a second reaction period canbegin. Alternately, the post-analysis conduit can serve simply toseparate the sample segment from the sensor. Within this post-analysisconduit is a single closeable valve which connects the air vent of thesensor conduit to the diaphragm air pump. When this valve closes, thesample is locked in the post analytical conduit and cannot be moved backto the sensor chip. There are several different design examples for thisvalve that are encompassed within the present invention. Some designsare activated mechanically while others activate on liquid contact.Other types of closeable valve that are encompassed by the presentinvention include, but are not limited to: a flexible flap held in anopen position by a soluble glue or a gelling polymer that dissolves orswells upon contact with a fluid or sample thus causing the flap toclose: and alternatively, in one specific embodiment, a thin layer of aporous paper or similar material interposed between a conduit and eitherthe waste chamber or ambient air such that the paper is permeable to airwhile dry but impermeable when wet. In the latter case it is notnecessary that the closeable valve be interposed between a conduit andthe waste chamber: the valve passes little to no liquid before closingand so the valve is appropriately placed when positioned between aconduit and the ambient air surrounding the cartridge. In practicalconstruction, a piece of filter paper is placed on an opening in thetape gasket in the fluid path to be controlled. Air can readily movethrough this media to allow fluid to be moved through the fluid path.When the fluid is pushed over this filter, the filter media becomesfilled with liquid and further motion through the fluid path is stopped.Once the filter become wet, significant pressures would be required tomove liquid through the pores of the filter. Air flow through the filteris also prevented because of the higher pressure required to push theliquid out of the filter. This valve embodiment requires very littleliquid to actuate the valve, and actuation occurs rapidly and reliably.Materials, their dimensions, porosity, wettability, swellingcharacteristics and related parameters are selected to provide for rapidclosure, within one second or more slowly, e.g., up to 60 seconds, afterfirst contacting the sample, depending on the specific desired closuretime.

Alternatively, the closeable valve is a mechanical valve. In thisembodiment, a latex diaphragm is placed in the bottom of the air bladderon top of a specially constructed well. The well contains two openingswhich fluidically connect the air vent to the sample conduit. As theanalyzer plunger pushes to the bottom of the air bladder, it presses onthis latex diaphragm which is adhesive backed and seals the connectionbetween the two holes. This blocks the sample's air vent, locking thesample in place.

Referring now to FIG. 6, which illustrates the schematic layout of animmunosensor cartridge, there are provided three pump means, 61-63.While these pumps have been described in terms of specific embodiments,it will be readily understood that any pump means capable of performingthe respective functions of pump means 61-63 may be used within thepresent invention. Thus, pump means 1, 61, must be capable of displacingthe sample from the sample holding chamber into the first conduit: pumpmeans 2, 62, must be capable of displacing fluid within the secondconduit: and pump means 3, 63, must be capable of inserting at least onesegment into the second conduit. Other types of pump which are envisagedin the present application include, but are not limited to, an air saccontacting a pneumatic means whereby pressure is applied to said airsac, a flexible diaphragm, a piston and cylinder, an electrodynamicpump, and a sonic pump. With reference to pump means 3, 63, the term“pump means” includes all methods by which one or more segments areinserted into the second conduit, such as a pneumatic means fordisplacing air from an air sac, a dry chemical that produces a gas whendissolved, or a plurality of electrolysis electrodes operably connectedto a current source. In a specific embodiment, the segment is producedusing a mechanical segment generating diaphragm that may have more thanone air bladder or chamber. The well 8 has a single opening whichconnects the inner diaphragm pump and the fluid filled conduit intowhich a segment is to be injected 20. The diaphragm can be segmented toproduce multiple segments, each injected in a specific location within afluid filled conduit.

In alternative embodiments, a segment is injected using a passivefeature. A well in the base of the cartridge is sealed by tape gasket.The tape gasket covering the well has two small holes on either end. Onehole is open while the other is covered with a filter material whichwets upon contact with a fluid. The well is filled with a loosehydrophilic material such as a cellulose fiber filter, paper filter orglass fiber filter. This hydrophilic material draws the liquid into thewell in the base via capillary action, displacing the air which wasformerly in the well. The air is expelled through the opening in thetape gasket creating a segment whose volume is determined by the volumeof the well and the volume of the loose hydrophilic material. The filterused to cover one of the inlets to the well in the base can be chosen tometer the rate at which the fluid fills the well and thereby control therate at which the segment is injected into the conduit in the cover.This passive feature permits any number of controlled segments to beinjected at specific locations within a fluid path and requires aminimum of space.

The present invention will be better understood with reference to thespecific embodiments set forth in the following examples.

EXAMPLE 1

Referring now to FIG. 7, which illustrates the principle of anamperometric immunoassay according to specific embodiments of thepresent invention for determination of troponin I (TnI), a marker ofcardiac function. A blood sample, for example, is introduced into thesample holding chamber of a cartridge of the present invention, and isamended by a conjugate molecule comprising alkaline phosphatase enzyme(AP) covalently attached to a polyclonal anti-troponin I antibody (aTnI)71. This conjugate specifically binds to the TnI, 70, in the bloodsample, producing a complex made up of TnI bound to the AP-aTnIconjugate. In a capture step, this complex binds to the capture aTnIantibody 72 attached on, or close to, the immunosensor. The sensor chiphas a conductivity sensor which is used to monitor when the samplereaches the sensor chip. The time of arrival of the fluid can be used todetect leaks within the cartridge: a delay in arrival signals a leak.The position of the sample segment within the sensor conduit can beactively controlled using the edge of the fluid as a marker. As thesample/air interface crosses the conductivity sensor, a precise signalis generated which can be used as a fluid marker from which controlledfluid excursions can be executed. The fluid segment is preferentiallyoscillated edge-to-edge over the sensor in order to present the entiresample to the sensor surface. A second reagent can be introduced in thesensor conduit beyond the sensor chip, which becomes homogenouslydistributed during the fluid oscillations.

The sensor chip contains a capture region or regions coated withantibodies for the analyte of interest. These capture regions aredefined by a hydrophobic ring of polyimide or anotherphotolithographically produced layer. A microdroplet or severalmicrodroplets (approximately 5-40 nanoliters in size) containingantibodies in some form, for example bound to latex microspheres, isdispensed on the surface of the sensor. The photodefined ring containsthis aqueous droplet allowing the antibody coated region to be localizedto a precision of a few microns. The capture region can be made from0.03 to roughly 2 square millimeters in size. The upper end of this sizeis limited by the size of the conduit and sensor in present embodiments,and is not a limitation of the invention.

Thus, the gold electrode 74 is coated with a biolayer 73 comprising acovalently attached anti-troponin I antibody, to which the TnI/AP-aTnIcomplex binds. AP is thereby immobilized close to the electrode inproportion to the amount of TnI initially present in the sample. Inaddition to specific binding, the enzyme-antibody conjugate may bindnon-specifically to the sensor. Non-specific binding provides abackground signal from the sensor that is undesirable and preferably isminimized. As described above, the rinsing protocols, and in particularthe use of segmented fluid to rinse the sensor, provide efficient meansto minimize this background signal. In a second step subsequent to therinsing step, a substrate 75 that is hydrolyzed by, for example,alkaline phosphatase to produce an electroactive product 76 is presentedto the sensor. In specific embodiments the substrate is comprised of aphosphorylated ferrocene or p-aminophenol. The amperometric electrode iseither clamped at a fixed electrochemical potential sufficient tooxidize or reduce a product of the hydrolyzed substrate but not thesubstrate directly, or the potential is swept one or more times throughan appropriate range. Optionally, a second electrode may be coated witha layer where the complex of TnI/AP-aTnI is made during manufacture, toact as a reference sensor or calibration means for the measurement.

In the present example, the sensor comprises two amperometric electrodeswhich are used to detect the enzymatically produced 4-aminophenol fromthe reaction of 4-aminophenylphosphate with the enzyme label alkalinephosphatase. The electrodes are preferably produced from gold surfacescoated with a photodefined layer of polyimide. Regularly spaced openingin the insulating polyimide layer define a grid of small gold electrodesat which the 4-aminophenol is oxidized in a 2 electron per moleculereaction. Sensor electrodes further comprise a biolayer, while referenceelectrodes can be constructed, for example, from gold electrodes lackinga biolayer, or from silver electrodes, or other suitable material.Different biolavers can provide each electrode with the ability to sensea different analyte.

H₂N—C₆H₄—OH→HN═C₆H₄═O+2H⁺+2e⁻

Substrates, such as p-aminophenol species, can be chosen such that the Eof the substrate and product differ substantially. Preferably, the E_(1/2) of the substrate is substantially higher than that of theproduct. When the condition is met, the product can be selectivelyelectrochemically measured in the presence of the substrate.

The size and spacing of the electrode play an important role indetermining the sensitivity and background signal. The importantparameters in the grid are the percentage of exposed metal and thespacing between the active electrodes. The position of the electrode canbe directly underneath the antibody capture region or offset from thecapture region by a controlled distance. The actual amperometric signalof the electrodes depends on the positioning of the sensors relative tothe antibody capture site and the motion of the fluid during theanalysis. A current at the electrode is recorded that depends upon theamount of electroactive product in the vicinity of the sensor.

The detection of alkaline phosphatase activity in this example relies ona measurement of the 4-aminophenol oxidation current. This is achievedat a potential of about +60 mV versus the Ag/AgCl ground chip. The exactform of detection used depends on the sensor configuration. In oneversion of the sensor, the array of gold microelectrodes is locateddirectly beneath the antibody capture region. When the analysis fluid ispulled over this sensor, enzyme located on the capture site converts the4-aminophenylphosphate to 4-aminophenol in an enzyme limited reaction.The concentration of the 4-aminophenylphosphate is selected to be inexcess, e.g., 10 times the Km value. The analysis solution is 0.1 M indiethanolamine, 1.0 M NaCl, buffered to a pH of 9.8. Additionally, theanalysis solution contains 0.5 mM MgCl which is a cofactor for theenzyme.

In another electrode geometry embodiment, the electrode is located a fewhundred microns away from the capture region. When a fresh segment ofanalysis fluid is pulled over the capture region, the enzyme productbuilds with no loss due to electrode reactions. After a time, thesolution is slowly pulled from the capture region over the detectorelectrode resulting in a current spike from which the enzyme activitycan be determined.

An important consideration in the sensitive detection of alkalinephosphatase activity is the non-4-aminophenol current associated withbackground oxidations and reductions occurring at the gold sensor. Goldsensors tend to give significant oxidation currents in basic buffers atthese potentials. The background current is largely dependent on thebuffer concentration, the area of the gold electrode (exposed area),surface pretreatments and the nature of the buffer used. Diethanolamineis a particularly good activating buffer for alkaline phosphatase. Atmolar concentrations, the enzymatic rate is increased by about threetimes over a non-activating buffer such as carbonate.

In alternative embodiments, the enzyme conjugated to an antibody orother analyte-binding molecule is urease, and the substrate is urea.Ammonium ions produced by the hydrolysis of urea are detected in thisembodiment by the use of an ammonium sensitive electrode.Ammonium-specific electrodes are well-known to those of skill in theart. A suitable microfabricated ammonium ion-selective electrode isdisclosed in U.S. Pat. No. 5,200,051, incorporated herein by reference.Other enzymes that react with a substrate to produce an ion are known inthe art, as are other ion sensors for use therewith. For example,phosphate produced from an alkaline phosphatase substrate can bedetected at a phosphate ion-selective electrode.

Referring now to FIG. 8, there is illustrated the construction of anembodiment of a microfabricated immunosensor. Preferably a planarnon-conducting substrate is provided, 80, onto which is deposited aconducting layer 81 by conventional means or microfabrication known tothose of skill in the art. The conducting material is preferably a noblemetal such as gold or platinum, although other unreactive metals such asiridium may also be used, as may non-metallic electrodes of graphite,conductive polymer, or other materials. An electrical connection 82 isalso provided. A biolayer 83 is deposited onto at least a portion of theelectrode. In the present disclosure, a biolayer means a porous layercomprising on its surface a sufficient amount of a molecule 84 that caneither bind to an analyte of interest, or respond to the presence ofsuch analyte by producing a change that is capable of measurement.Optionally, a permselective screening layer may be interposed betweenthe electrode and the biolayer to screen electrochemical interferents asdescribed in U.S. Pat. No. 5,200,051.

In specific embodiments, a biolayer is constructed from latex beads ofspecific diameter in the range of about 0.001 to 50 microns. The beadsare modified by covalent attachment of any suitable molecule consistentwith the above definition of a biolayer. Many methods of attachmentexist in the art, including providing amine reactiveN-hydroxysuccinimide ester groups for the facile coupling of lysine orN-terminal amine groups of proteins. In specific embodiments, thebiomolecule is chosen from among ionophores, cofactors, polypeptides,proteins, glycopeptides, enzymes, immunoglobulins, antibodies, antigens,lectins, neurochemical receptors, oligonucleotides, polynucleotides,DNA, RNA, or suitable mixtures. In most specific embodiments, thebiomolecule is an antibody selected to bind one or more of humanchorionic gonadotrophin, troponin I, troponin T, troponin C, a troponincomplex, creatine kinase, creatine kinase subunit M, creatine kinasesubunit B, myoglobin, myosin light chain, or modified fragments ofthese. Such modified fragments are generated by oxidation, reduction,deletion, addition or modification of at least one amino acid, includingchemical modification with a natural moiety or with a synthetic moiety.Preferably, the biomolecule binds to the analyte specifically and has anaffinity constant for binding analyte ligand of about 10⁷ to 10¹⁵ M⁻¹.

In one embodiment, the biolayer, comprising beads having surfaces thatare covalently modified by a suitable molecule, is affixed to the sensorby the following method. A microdispensing needle is used to depositonto the sensor surface a small droplet, preferably about 0.4 nl, of asuspension of modified beads. The droplet is permitted to dry, whichresults in a coating of the beads on the surface that resistsdisplacement during use.

In addition to immunosensors in which the biolayer is in a fixedposition relative to an amperometric sensor, the present invention alsoenvisages embodiments in which the biolaver is coated upon particlesthat are mobile. The cartridge can contain mobile microparticles capableof interacting with an analyte, for example magnetic particles that arelocalized to an amperometric electrode subsequent to a capture step,whereby magnetic forces are used to concentrate the particles at theelectrode for measurement. One advantage of mobile microparticles in thepresent invention is that their motion in the sample or fluidaccelerates binding reactions, making the capture step of the assayfaster. For embodiments using non-magnetic mobile microparticles, aporous filter is used to trap the beads at the electrode.

Referring now to FIG. 9, there is illustrated a mask design for severalelectrodes upon a single substrate. By masking and etching techniques,independent electrodes and leads can be deposited. Thus, a plurality ofimmunosensors, 94 and 96, and conductimetric sensors, 90 and 92, areprovided in a compact area at low cost, together with their respectiveconnecting pads, 91, 93, 95, and 97, for effecting electrical connectionto the reading apparatus. In principle, a very large array of sensorscan be assembled in this way, each sensitive to a different analyte oracting as a control sensor.

Specifically, immunosensors are prepared as follows. Silicon wafers arethermally oxidized to form approximately a 1 micron insulating oxidelayer. A titanium/tungsten layer is sputtered onto the oxide layer to apreferable thickness of between 100-1000 Angstroms, followed by a layerof gold that is most preferably 800 Angstroms thick. Next, a photoresistis spun onto the wafer and is dried and baked appropriately. The surfaceis then exposed using a contact mask, such as a mask corresponding tothat illustrated in FIG. 9. The latent image is developed, and the waferis exposed to a gold-etchant. The patterned gold layer is coated with aphotodefinable polyimide, suitably baked, exposed using a contact mask,developed, cleaned in an O₂ plasma, and preferably imidized at 350° C.for 5 hours. The surface is then printed with antibody-coated particles.Droplets, preferably of about 0.4 nl volume and containing 2% solidcontent in deionized water, are deposited onto the sensor region and aredried in place by air drying. Optionally, an antibody stabilizationreagent (e.g., Stabilicoat, obtained from SurModica Corp.) is overcoatedonto the sensor.

Drying the particles causes them to adhere to the surface in a mannerthat prevents dissolution in either sample or fluid containing asubstrate. This method provides a reliable and reproducibleimmobilization process suitable for manufacturing sensor chips in highvolume.

Referring now to FIG. 10, there are illustrated results obtained foranalysis of samples containing 0 or 50 miU/mL, human chorionicgonadotrophin (HCG) and an HCG -sensitive amperometric immunosensor. Attime 100, a solution containing a p-aminophenol phosphate is supplied toa sensor which is previously treated with HCG and an anti-HCG polyclonalantibody conjugated to alkaline phosphatase. As the substrate ishydrolyzed by alkaline phosphatase, a current increases to a maximum101, and thereafter declines 102, as substrate within the diffusionvolume of the sensor is depleted and oxidized p-aminophenol accumulates.Good reproducibility is obtained between sensors, as shown by the outputsignal characteristics of individual single-use sensors. In operation,displacement of the fluid containing the enzyme substrate provides freshsubstrate to the electrode surface, and also removes products, so thatmultiple readings are easily obtained for a single sample. In analternative embodiment, the signal at the electrode is augmented byenzymatic regeneration of the electroactive species in the vicinity ofthe electrode. In a specific embodiment, a phosphorylated ferrocene isused as the substrate for alkaline phosphatase attached to the antibody.Hydrolysis yields a ferrocene product, which is oxidized and detected atthe electrode. In a second step, glucose oxidase enzyme and glucose areused to re-reduce the electrochemically oxidized ferrocene, with aconsequent increase in the current and detection sensitivity. Referringnow to FIG. 13, an electrode 130 oxidizes or reduces the electroactiveproduct 132 of alkaline phosphatase immobilized as a complex 131 on orclose to the electrode surface. In a second step, the electroactivespecies 132 is regenerated from the product 133 by the catalytic actionof enzyme 134. This cycling reaction increases the concentration ofelectroactive species 132 in proximity to the electrode surface 130, andthereby increases the current recorded at the electrode.

Referring now to FIG. 11, there is shown dose-response results obtainedusing HCG and an HCG-responsive amperometric immunosensor. Amounts ofHCG equivalent to 0 to 50 miU/mL are allowed to bind to the immobilizedantibody attached to the electrode, as in FIG. 10. Referring now to FIG.12, good linearity, 121 of the response of the peak sensor current withincreasing HCG is found. Thus, it is demonstrated that this embodimentcan precisely and rapidly quantify HCG in a sample.

EXAMPLE 2 Method of Use a Cartridge of Claim 1

In a first cartridge embodiment, one exemplary analyte assay protocolusing a cartridge of claim 1 is described. An unmetered fluid sample isintroduced into sample chamber 34 of a cartridge according to claim 1,through sample entry port 4. Capillary stop 25 prevents passage of thesample into conduit 11 at this stage, and conduit 34 is filled with thesample. Lid 2 is closed to prevent leakage of the sample from thecartridge. The cartridge is then inserted into a reading apparatus, suchas that disclosed in U.S. Pat. No. 5,821,399 to Zelin, which is herebyincorporated by reference. Insertion of the cartridge into a readingapparatus activates the mechanism which punctures a fluid-containingpackage located at 42 when the package is pressed against spike 38.Fluid is thereby expelled into the second conduit, arriving in sequenceat 39, 20, 12 and 11. The constriction at 12 prevents further movementof fluid because residual hydrostatic pressure is dissipated by the flowof fluid via second conduit portion 11 into the waste chamber 44. In asecond step, operation of a pump means applies pressure to air-bladder43, forcing air through conduit 40, through cutaways 17 and 18, and intoconduit 34 at a predetermined location 27. Capillary stop 25 andlocation 27 delimit a metered portion of the original sample. While thesample is within sample chamber 34, it is optionally amended with acompound or compounds present initially as a dry coating on the innersurface of the chamber. The metered portion of the sample is thenexpelled through the capillary stop by air pressure produced within airbladder 43. The sample passes into conduit 15 and into contact with theanalyte sensor or sensors located within cutaway 35.

In embodiments employing an immunosensor located within cutout 35, thesample is amended prior to arriving at the sensor by, for example, anenzyme-antibody conjugate. An antibody that binds the analyte ofinterest is covalently attached to an enzyme that can generate a redoxactive substance close to an amperometric electrode. In specificembodiments, the enzyme may be alkaline phosphatase, which hydrolyzescertain organophosphate compounds, such as derivatives of p-aminophenolthat liberate redox-active compounds when hydrolyzed. However, anyenzyme capable of producing, destroying, or altering any compound thatmay be detected by a sensor may be employed in conjunction with amatching sensor. For example, antibody-urease conjugate may be usedtogether with an ammonium sensor. Thus, the enzyme-antibody conjugate orconjugates amends the sample and binds to the analyte of interest. Theimmunosensor can comprise immobilized antibody that binds to an analyteof interest. When the amended sample passes over the immunosensor, theanalyte of interest binds to the sensor, together with antibody-enzymeconjugate to which it is attached.

To promote efficient binding of the analyte to the sensor, the samplecontaining the analyte is optionally passed repeatedly over the sensorin an oscillatory motion. Preferably, an oscillation frequency ofbetween about 0.2 and 2 Hz is used, most preferably 0.7 Hz. Thus enzymeis brought into close proximity to the amperometric electrode surface inproportion to the amount of analyte present in the sample.

Once an opportunity for the analyte/enzyme-antibody conjugate complex tobind to the immunosensor has been provided, the sample is ejected byfurther pressure applied to air bladder 43, and the sample passes towaste chamber 44.

A wash step next removes non-specifically bound enzyme-conjugate fromthe sensor chamber. Fluid in the second conduct is moved by a pump means43, into contact with the sensors. The analysis fluid is pulled slowlyuntil the first air segment is detected at a conductivity sensor.

The air segment or segment can be produced within a conduit by anysuitable means, including but not limited to, passive means, as shown inFIG. 14 and described below: active means including a transient loweringof the pressure within a conduit using pump means whereby air is drawninto the conduit through a flap or valve: or by dissolving a compoundpre-positioned within a conduit that liberates a gas upon contactingfluid in the conduit, where such compound may be a carbonate,bicarbonate or the like. This segment is extremely effective at clearingthe sample-contaminated fluid from conduit 15. The efficiency of therinsing of the sensor region is greatly enhanced by the introduction ofone or more air segments into the second conduit as described. Theleading and/or trailing edges of air segments are passed one or moretimes over the sensors to rinse and resuspend extraneous material thatmay have been deposited from the sample. Extraneous material includesany material other than specifically bound analyte oranalyte/antibody-enzyme conjugate complex. However, it is an object ofthe invention that the rinsing is not sufficiently protracted orvigorous as to promote dissociation of specifically bound analyte oranalyte/antibody-enzyme conjugate complex from the sensor.

A second advantage of introducing air segments into the fluid is tosegment the fluid. For example, after a first segment of the fluid isused to rinse a sensor, a second segment is then placed over the sensorwith minimal mixing of the two segments. This feature further reducesbackground signal from the sensor by more efficiently removing unboundantibody-enzyme conjugate. After the front edge washing, the analysisfluid is pulled slowly until the first air segment is detected at aconductivity sensor. This segment is extremely effective at clearing thesample-contaminated fluid which was mixed in with the first analysisfluid sample.

A second advantage of introducing air segments into conduit two is tosegment the fluid. For example, after a first segment of the fluid isused to rinse a sensor, a second segment is then placed over the sensorwith minimal mixing of the two segments. This feature further reducesbackground signal from the sensor by more efficiently removing unboundantibody-enzyme conjugate.

For measurement, a new portion of fluid is placed over the sensors, andthe current or potential, as appropriate to the mode of operation, isrecorded as a function of time.

EXAMPLE 3 Method of Use of the Cartridge of Claim 2

The cartridge of claim 2 comprises all the elements of the cartridge ofclaim 1 together with a closeable valve, preferably located between thesensor chamber and the waste chamber. The method of use of the cartridgeof claim 2 is herein illustrated by a specific embodiment in which theconcentration of HCG is determined within a blood sample, which isintroduced into the sample chamber of said cartridge. In the followingtime sequence, time zero (t=0) represents the time at which thecartridge is inserted into the cartridge reading device. Times are givenin minutes. Between t=0 and t=1.5, the cartridge reading device makeselectrical contact with the sensors through pads 91, 93, 95, and 97, andperforms certain diagnostic tests. Insertion of the cartridge perforatesthe foil pouch introducing fluid into the second conduit as previouslydescribed. The diagnostic tests determine whether fluid or sample ispresent in the conduits using the conductivity electrodes: determinewhether electrical short circuits are present in the electrodes: andensure that the sensor and ground electrodes are thermally equilibratedto, preferably, 37° C. prior to the analyte determination.

Between t=1.5 and t=6.75, a metered portion of the sample, preferablybetween 4 and 200 μl, more preferably between 4 and 20 μl, and mostpreferably 7 μl, is used to contact the sensor as described in EXAMPLE2. The edges defining the forward and trailing edges of the sample arereciprocally moved over the sensor region at a frequency that ispreferably between 0.2 to 2.0 Hz, and is most preferably 0.7 Hz. Duringthis time, the enzyme-antibody conjugate dissolves within the sample, aspreviously described. The amount of enzyme-antibody conjugate that iscoated onto the conduit is selected to yield a concentration whendissolved that is preferably higher than the highest anticipated HCGconcentration, and is most preferably six times higher than the highestanticipated HCG concentration in the sample.

Between t=6.75 and t=10.0 the sample is moved into the waste chamber viacloseable valve 41, wetting the closeable valve and causing it to closeas previously described. The seal created by the closing of the valvepermits the first pump means to be used to control motion of fluid fromconduit 11 to conduit 15. After the valve closes and the any remainingsample is locked in the post analysis conduit, the analyzer plungerretracts from the flexible diaphragm of the pump mean creating a partialvacuum in the sensor conduit. This forces the analysis fluid through thesmall hole in the tape gasket 31 and into a short transecting conduit inthe base, 13, 14. The analysis fluid is pulled further and the frontedge of the analysis fluid is oscillated across the surface of thesensor chip in order to shear the sample near the walls of the conduit.A conductivity sensor on the sensor chip is used to control thisprocess. The efficiency of the process is monitored using theamperometric sensors through the removal of unbound enzyme-antibodyconjugate which enhances the oxidation current measured at the electrodewhen the enzyme substrate, 4-aminophenyl phosphate is also present. Theamperometric electrodes are polarized to 0.06 V versus the silverchloride reference-ground electrode. In this embodiment, the fluid iscomposed of a 0.1 M diethanolamine buffer, pH 9.8, with 1 mM MgCl₂, 1.0M NaCl, 10 mM 4-aminophenylphosphate, and 10μM NaI. The efficiency ofthe wash is optimally further enhanced by introduction into the fluid ofone or more segments that segment the fluid within the conduit aspreviously described. The air segment may be introduced by either activeor passive means. Referring now to FIG. 14, there is illustrated theconstruction of a specific means for passively introducing an airsegment into said fluid. Within the base of the immunosensor is recess140 comprising a tapered portion 141 and a cylindrical portion that areconnected. The tapered portion is in fluid connection with a hole 142 ofsimilar diameter in the tape gasket (FIG. 3) that separates the base(FIG. 4) and cover (FIGS. 1 and 2) of the assembled immunosensorcartridge. The recess contains an absorbent material that, upon contactwith fluid, withdraws a small quantity of fluid from a conduit therebypassively introducing an air segment into the conduit. The volume of therecess and the amount and type of material within it may be adjusted tocontrol the size of the air segment introduced. Specific materialsinclude, but are not limited to, glass filter, a laminate comprising a 3micron Versapor filter bonded by sucrose to a 60% viscose chiffon layer.

Fluid is forcibly moved towards sensor chip by the partial vacuumgenerated by reducing the mechanical pressure exerted upon paddle 6,causing the “T” region of the sensor channel in the vicinity of thetransecting conduit to fill with analysis fluid. The T region of thesensor channel optionally has a higher channel height resulting ameniscus with a smaller radius of curvature. Further away from the Tregion towards the post-analytical conduit, the conduit height isoptionally smaller. The analysis fluid passively flows from the T regiontowards this low conduit height region washing the conduit walls. Thispassive leak allows further effective washing of the T region using aminimal volume of fluid.

In this simple embodiment, the fluid located within the second conduitcontains a substrate for the enzyme. In other embodiments, amendment ofthe fluid using dried substrate within the second conduit may be used.

Following the positioning of a final segment of fluid over the sensor,measurement of the sensor response is recorded and the concentration ofanalyte determined as described for EXAMPLE 2. Specifically, at leastone sensor reading of a sample is made by rapidly placing over thesensor a fresh portion of fluid containing a substrate for the enzyme.Rapid displacement both rinses away product previously formed, andprovides now substrate to the electrode. Repetitive signals are averagedto produce a measurement of higher precision, and also to obtain abetter statistical average of the baseline, represented by the currentimmediately following replacement of the solution over the sensor.

B. Preferred Embodiment Cartridge Construction and Operation:

Referring now to FIG. 15, there is shown a top view of the preferredembodiment of an immunosensor cartridge. The preferred embodimentdiffers from the specific embodiments of Section A in certain featuresand methods of use that are advantageous for the rapid, reproducible,and inexpensive determination of analytes. The preferred embodimentcartridge shares many features in common with the specific embodimentcartridges described above, and are therefore described with emphasis onspecific differences. One skilled in the art to which the inventionpertains will readily appreciate from the combined descriptions ofSections A and B the construction and use of the preferred embodiment.

The preferred embodiment cartridge 150 comprises a base and a topportion, preferably constructed of a plastic. The two portions areconnected by a thin, adhesive gasket or thin pliable film. As inprevious embodiments, the assembled cartridge comprises a sample chamber151 into which a sample containing an analyte of interest is introducedvia a sample inlet 152. A metered portion of the sample is delivered tothe sensor chip 153, via the sample conduit 154 (first conduit) asbefore by the combined action of a capillary stop 152, preferably formedby a 0.012″ laser cut hole in the gasket or film that connects the twoportions of the cartridge, and an entry point 155 located at apredetermined point within the sample chamber whereby air introduced bythe action of a pump means, such as a paddle pushing upon a samplediaphragm 156. After contacting the sensor to permit binding to occur,the sample is moved to vent 157, which contains a wicking material thatabsorbs the sample and thereby seals the vent closed to the furtherpassage of liquid or air. The wicking material is preferably a cottonfiber material, a cellulose material, or other hydrophilic materialhaving pores. It is important in the present application that thematerial is sufficiently absorbent (i.e., possesses sufficient wickingspeed) that the valve closes within a time period that is commensuratewith the subsequent withdrawal of the sample diaphragm actuating meansdescribed below, so that sample is not subsequently drawn back into theregion of the sensor chip.

As in the specific embodiments, there is provided a wash conduit (secondconduit) 158, connected at one end to a vent 159 and at the other end tothe sample conduit at a point 160 of the sample conduit that is locatedbetween vent 157 and sensor chip 153. Upon insertion of the cartridgeinto a reading apparatus, a fluid is introduced into conduit 158.Preferably, the fluid is present initially within a foil pouch 161 thatis punctured by a pin when an actuating means applies pressure upon thepouch. There is also provided a short conduit 162 that connects thefluid to conduit 154 via a small opening in the gasket 163. A secondcapillary stop initially prevents the fluid from reaching capillary stop160, so that the fluid is retained within conduit 158.

After vent 157 has closed, the pump means is actuated, creating alowered pressure within conduit 154. Air vent 164, preferably comprisinga small flap cut in the gasket or a membrane that vibrates to provide anintermittent air stream, provides a means for air to enter conduit 158via a second vent 165. The second vent 165 preferably also containswicking material capable of closing the vent if wetted, which permitssubsequent depression of sample diaphragm 156 to close vent 165, ifrequired. Simultaneously with the actuation of sample diaphragm 156,fluid is drawn from conduit 158, through capillary stop 160, intoconduit 154. Because the flow of fluid is interrupted by air enteringvent 164, at least one air segment (a segment or stream of segments) isintroduced.

Further withdrawal of sample diaphragm 156 draws the liquid containingat least one air segment back across the sensing surface of sensor chip153. The presence of air-liquid boundaries within the liquid enhancesthe rinsing of the sensor chip surface to remove remaining sample.Preferably, the movement of the sample diaphragm 156 is controlled inconjunction with signals received from the conductivity electrodeshoused within the sensor chip adjacent to the analyte sensors. In thisway, the presence of liquid over the sensor is detected, and multiplereadings can be performed by movement of the fluid in discrete steps.

It is advantageous in this preferred embodiment to perform analytemeasurements when only a thin film of fluid coats the sensors, groundchip 165, and a contiguous portion of the wall of conduit 154 betweenthe sensors and ground electrode. A suitable film is obtained bywithdrawing fluid by operation of the sample diaphragm 156, until theconductimetric sensor located next to the sensor indicates that bulkfluid is no longer present in that region of conduit 154. It has beenfound that measurement can be performed at very low (nA) currents, thepotential drop that results from increased resistance of a thin filmbetween ground chip and sensor chip (compared to bulk fluid), is notsignificant.

The ground chip 165 is preferably silver/silver chloride. It isadvantageous, to avoid air segments, which easily form upon therelatively hydrophobic silver chloride surface, to pattern the groundchip as small regions of silver/silver chloride interspersed with morehydrophilic regions, such as a surface of silicon dioxide. Thus, apreferred ground electrode configuration comprises an array ofsilver/silver chloride squares densely arranged and interspersed withsilicon dioxide. There is a further advantage in the avoidance ofunintentional segments if the regions of silver/silver chloride aresomewhat recessed.

Referring now to FIG. 16, there is shown a schematic view of thefluidics of the preferred embodiment of an immunosensor cartridge.Regions R1-R7 represent specific regions of the conduits associated withspecific operational functions. Thus R1 represents the sample chamber:R2 the sample conduit whereby a metered portion of the sample istransferred to the capture region, and in which the sample is optionallyamended with a substance coated upon the walls of the conduit: R3represents the capture region, which houses the conductimetric andanalyte sensors: R4 and R5 represent portions of the first conduit thatare optionally used for further amendment of fluids with substancescoated onto the conduit wall, whereby more complex assay schemes areachieved: R6 represents the portion of the second conduit into whichfluid is introduced upon insertion of the cartridge into a readingapparatus: R7 comprises a portion of the conduit located betweencapillary stops 160 and 166, in which further amendment can occur: andR8 represents the portion of conduit 154 located between point 160 andvent 157, and which can further be used to amend liquids containedwithin.

EXAMPLE 4 Coordination of Fluidics and Analyte Measurement in aCartridge of the Preferred Embodiment

The use of the preferred embodiment immunocartridge is illustrate inthis example. In the analysis sequence, a user places a sample into thecartridge, places the cartridge into the analyzer and in 1 to 20minutes, a quantitative measurement of one or more analytes isperformed. Herein is a non-limiting example of a sequence of events thatoccur during the analysis:

1) A 25 to 50 uL sample is introduced in the sample inlet 167 and fillsto a capillary stop 151 formed by a 0.012″ laser cut hole in theadhesive tape holding the cover and base components together. The userrotates a latex rubber disk mounted on a snap flap to close the sampleinlet 167 and places the cartridge into the analyzer.

2) The analyzer makes contact with the cartridge, and a motor drivenplunger presses onto the foil pouch 161 forcing the wash/analysis fluidout into a central conduit 158.

3) A separate motor driven plunger contacts the sample diaphragm 156pushing a measured segment of the sample along the sample conduit (fromreagent region R1 to R2). The sample is detected at the sensor chip 153via the conductivity sensors. The sensor chip is located in captureregion R3.

4) The sample is oscillated by means of the sample diaphragm 156 betweenR2 and R5 in a predetermined and controlled fashion for a controlledtime to promote binding to the sensor.

5) The sample is pushed towards the waste region of the cartridge (R8)and comes in contact with a passive pump 157 in the form of a celluloseor similar absorbent wick. The action of wetting this wick seals thewick to air flow thus eliminating its ability to vent excess pressuregenerated by the sample diaphragm 156. The active vent becomes the“controlled air vent” of FIG. 16.

6) Rapid evacuation of the sample conduit (effected by withdrawing themotor driven plunger from the sample diaphragm 156) forces a mixture ofair (from the vent) and wash/analysis fluid from the second conduit tomove into the inlet located between R5 and R4 in FIG. 16. By repeatingthe rapid evacuation of the sample conduit, a series of air separatedfluid segments are generated which are pulled across the sensor chiptowards the sample inlet (from R4 to R3 to R2 and R1). This washes thesensor free of excess reagents and wets the sensor with reagentsappropriate for the analysis. The wash/analysis fluid which originatesin the foil pouch can be further amended by addition of reagents in R7and R6 within the central wash/analysis fluid conduit.

7) The wash/analysis fluid segment is drawn at a slower speed towardsthe sample inlet to yield a sensor chip which contains only a thin layerof the analysis fluid. The electrochemical analysis is performed at thispoint. The preferred method of analysis is amperometry but potentiometryor impedance detection is also used.

8) And the mechanism retracts allowing the cartridge to be removed fromthe analyzer.

Referring now to FIG. 17, there is illustrated an electrical signal 170representing the position of the electric motor actuating the samplediaphragm 156, the response 171 of the conductimetric electrode, and theelectrochemical response 172 of a amperometric immunosensor. In the timeperiod prior to 40 seconds after initiation of the immunoassay 173, themotor depresses the diaphragm, which pushes the sample into the captureregion and over the conductimetric sensor. Thus, after about 10 seconds,the conductivity rises to a steady value representative of samplefilling the portion of the conduit containing the conductimetric sensor.During this period the valve is sealed by contact with the sample.Between 40 seconds and about 63 seconds, the motor position is steppedback in increments 174, creating a periodic fluctuation in pressure,which draws an air-segmented portion of wash fluid over the sensor.During this period, fluctuations 175 in the immunoassay sensor are seen.At 177, the conductimetric response indicates that the wash fluid, whichcontains substrate, covers the conductimetric sensor. As the fluid isdrawn slowly over the sensor, a potential is applied (in this example,every five seconds, for 2.5 second periods) to the sensor, resulting inresponse 176, which indicates the presence of analyte bound to thesensor.

The invention described and disclosed herein has numerous benefits andadvantages compared to previous devices. These benefits and advantagesinclude, but are not limited to ease of use, the automation of most ifnot all steps of the analysis, which eliminates user included error inthe analysis.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes can be made withoutdeparting from the spirit of the present invention. Accordingly, it isintended that the scope of the present invention be limited solely bythe scope of the following claims.

1-20. (canceled)
 21. A method of measuring an amount of an analyte usingan electrochemical assay in a conduit comprising a sensor, wherein saidsensor comprises an electrode having a surface layer of immobilizedantibody that binds said analyte, and a counter/reference electrodedisposed within said conduit, said method comprising: contacting saidsensor with a liquid sample containing said analyte; contacting saidsensor with an enzyme-labeled antibody capable of binding said analyte,whereby a complex of immobilized antibody, analyte and labeled antibodyis formed; contacting said sensor with a solution comprising a substratefor said enzyme and at least one air segment to remove unbound analyteand labeled antibody from a region of the sensor; removing substantiallyall said solution from said sensor while retaining a portion of saidsolution over said electrode, said counter/reference electrode, and acontiguous portion of wall of the conduit connecting said electrodes;and detecting he a product of a reaction between said enzyme-labeledantibody and said substrate using said sensor, whereby the amount ofsaid analyte in the liquid sample is measured.
 22. The method of claim21, wherein the sample comprises the enzyme-labeled antibody dissolvedtherein.
 23. The method of claim 21, wherein the enzyme-labeled antibodyis subsequently delivered in a liquid to the sensor that is not thesample, prior to delivery of said solution.
 24. The method of claim 21,wherein the retained portion of said solution comprises a predeterminedvolume.
 25. The method of claim 21, wherein at least a portion of thewall of the conduit is treated to reduce non-specific binding of theenzyme-labeled antibody.
 26. The method of claim 21, wherein the atleast one air segment is formed by evacuation of the conduit.
 27. Themethod of claim 21, further comprising: displacing the liquid samplefrom the sensor into a waste region prior to the contacting the sensorwith the solution; and providing the solution in another conduit,wherein the contacting the sensor with the solution comprises displacingthe solution from the another conduit into contact with the sensor. 28.The method of claim 27, wherein the displacing the liquid sample intothe waste region closes a first vent.
 29. The method of claim 28,wherein the at least one air segment is formed by forcing air through asecond vent into the conduit.
 30. The method of claim 29, wherein: thedisplacing the solution further comprises actuating a pump to create alower pressure in the conduit; and the actuating the pump occurssubsequent to the closing the first vent.
 31. The method of claim 30,wherein: the second vent comprises a membrane that vibrates as the airis forced through the second vent providing an intermittent air stream;and the intermittent air stream interrupts displacing the solution fromthe another conduit into contact with the sensor, and causes the formingthe at least one air segment.
 32. A method of measuring an amount of ananalyte in a sample, the method comprising: displacing the sample from asample inlet to a first conduit comprising a sensor; contacting thesample with the sensor; contacting an enzyme-labeled antibody with thesensor, wherein the enzyme-labeled antibody is configured to bind theanalyte in the sample; displacing the sample from the sensor into awaste region; providing a solution in a second conduit, wherein thesolution comprises a substrate for the enzyme-labeled antibody;displacing the solution from the second conduit into the first conduit,wherein the displacing the solution comprises forming at least one airsegment in the solution; contacting the solution and the at least oneair segment with the sensor to remove unbound analyte and labeledantibody from a region of the sensor; and detecting a product of areaction between the enzyme-labeled antibody and the substrate using thesensor.
 33. The method of claim 32, wherein the displacing the sampleinto the waste region closes a first vent.
 34. The method of claim 33,wherein the forming the at least one air segment comprises forcing airthrough a second vent into the first conduit.
 35. The method of claim33, wherein the closing the first vent prevents the first vent fromventing excess pressure and subsequently displacing the sample back intothe first conduit.
 36. The method of claim 33, wherein: the displacingthe solution further comprises actuation of a pump to create a lowerpressure in the first conduit, the lower pressure draws the solutionfrom the second conduit through a capillary stop and into the firstconduit; and the actuation of the pump occurs subsequent to the closingthe first vent.
 37. The method of claim 34, wherein: the second ventcomprises a membrane that vibrates as the air is forced through thesecond vent providing an intermittent air stream; and the intermittentair stream interrupts the displacing the solution from the secondconduit into the first conduit, and causes the forming the at least oneair segment.
 38. The method of claim 32, further comprising displacingthe solution and the at least one air segment from the sensor towardsthe sample inlet such that only a thin layer of the solution remains onthe sensor.
 39. The method of claim 32, wherein the sample comprises theenzyme-labeled antibody dissolved therein.
 40. The method of claim 32,wherein the contacting the enzyme-labeled antibody with the sensoroccurs subsequent to the contacting the sample with the sensor and priorto the contacting the solution and the at least one air segment with thesensor.